System and Method for Antibiotic Delivery Using Single-Walled Carbon Nanotubes

ABSTRACT

A new route is shown for antibiotic delivery in fighting drug resistant infections. Nanotubes and antibiotics and complexed non-covalently, with no chemical bonding, but through adsorption of antibiotics onto the nanotube surface governed by sufficiently strong molecular attraction between hydrophobic systems of the two. This allows the antibiotics to be freed from the nanotubes more easily as they reach the cell membrane. When antibiotics are introduced with nanotubes in this manner, bacterial resistance is mitigated by nanotube transport potentially into the membrane of the bacteria. Nanotubes used in this way help to overcome antibiotic resistance.

CROSS-REFERENCE TO RELATED APPLICATION

Any and all applications for which a foreign or domestic priority claimis identified in the Application Data Sheet (either filed with thepresent application or subsequently amended) are hereby incorporated byreference under 37 CFR § 1.57. The present application cross-referencesand claims priority to U.S. Provisional Patent Application Ser. No.62/670,949, filed May 14, 2018, and U.S. application Ser. No.16/366,007, filed on Mar. 27, 2019, and these applications are hereinincorporated by reference as examples. The present application is acontinuation of U.S. application Ser. No. 16/366,007, filed on Mar. 27,2019.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates generally to the field of carbon nanotubesand to methods for using these nanotubes in therapeutic antibioticdelivery systems and in circumventing antibiotic resistance.

Description of the Prior Art

Infectious disease remains one of the leading causes of death worldwide.Although antibiotics revolutionized medicine in the 20^(th) century,overuse and misuse during the past 50 years has allowed for rapidemergence of drug resistance among numerous pathogens. In turn, therehas been a tremendous decline in treatment efficacy for commoninfections as well as an increase in severe opportunistic infectionscaused by bacteria that now have resistance to multiple antibiotics.Multidrug resistant (MDR) bacteria including Staphylococcus aureus, E.coli, Pseudomonas aeruginosa, and Klebsiella pneumonia pose anever-increasing threat as they are associated with severe nosocomialinfections in healthcare settings, and, more recently, have gainedprominence as the causative agents of community-acquired infections. Inaddition to the aforementioned bacteria, Staphylococcus epidermidis hasgained prominence as an MDR, opportunistic pathogen.

This bacterium has historically been viewed as being a harmless,commensal species but recent evidence suggests that its role in neonatalmorbidity has been grossly underestimated. Furthermore, it has becomeone of the most common causative agents of nosocomial infections. Todate, MDR pathogens have exhibited resistance towards all existingclasses of antibiotics. Escalating drug resistance has been facilitatedby the ease of transmission of resistance genes among different types ofbacteria for several types of antibiotics, further underscoring the needto address this global health crisis. Without significant advancementsto current standards of care, it is estimated that antibiotic-resistantbacterial infections will become the leading cause of death by 2050,outpacing cancer-related deaths worldwide.

Significant efforts are underway in order to thwart mounting drugresistance. As an example, the development of inhibitory compounds todisable efflux pumps are a major focus. Efflux pumps are used bybacteria to bind to and pump antibiotics out of the bacterial cell,thereby minimizing the intracellular concentration of drugs andrendering them ineffective. However, toxic side effects have preventedefflux pump inhibitors from being brought to the clinic to date.Separately, there is tremendous focus on developing methods in order tostimulate immune-mediated clearance of pathogens (and cancer cells). Itis thought that immunotherapeutics will offer enhanced treatmentefficacy while also minimizing the risk for adverse side effects.Moreover, by targeting host molecules as opposed to bacterial targets,immunotherapeutics would mitigate developing drug resistance.Additionally, there is significant emphasis on the use of antibioticpotentiators in order to restore efficacy for existing drug compounds.

In spite of the above areas in which advances have been made, a needexists for drug delivery systems which would allow for more effectivedrug delivery and uptake, thereby overcoming limitations with existingdrug therapies.

SUMMARY OF THE INVENTION

The present invention involves the use of single-walled carbon nanotubes(SWCNTs) complexed to antibiotics to facilitate delivery and uptake,imaging, and enhanced antibacterial activity. To date, the use of SWCNTscomplexed with different antibiotics has been demonstrated to show theincreased antibacterial activity against drug-resistant Staphylococcusepidermidis. The proposed use of SWCNTs, as described herein, incombination with existing antibiotics, could allow for enhanced deliveryand drug efficacy, while also allowing for fluorescence-based trackingof transported therapeutics.

The improved systems and techniques of the invention thus involve aparticular type of antibiotic delivery system which is based uponsingle-walled carbon nanotubes (SWCNTs). It is known that thecharacteristics of SWCNTs indicate that they are well suited forcellular internalization, exhibit low cytotoxicity when formulated withdrug compounds, and, that a significant amount of SWCNTs can be loadedonto a target cell. Collectively, these characteristics make SWCNTsideally suited for drug delivery. As an added benefit, it is indicatedthat SWCNTs can be used for fluorescence imaging to track the locationof drug therapeutics in cells and tissues with low backgroundinterference.

Given these features, the present invention deals with the developmentof improved antibiotic delivery systems of the type comprised of SWCNTscomplexed with antibiotics. In the case of the present invention, theSWCNTs were non-covalently complexed with doxycycline and methicillin inseparate experiments and dispersed in water. In each case, SWCNTs withantibiotics attached to their surface correlated with enhancedantibacterial activity against Staphylococcus epidermidis. Multiplesensitivity assays were performed to confirm enhanced antibacterialefficacy by the SWCNTs complexed with antibiotics. Notably, the resultsshow a 68% increase in bacterial colony inhibition for SWCNT/doxycyclineand 40-fold improvement in bacterial colony inhibition for*SWCNT/methicillin, in which S. epidermidis is initially resistant tomethicillin. These findings support the ability of SWCNTs to serve aseffective antibiotic delivery systems for multiples types of drugs.Moreover, the invention demonstrates the potential to bypass antibioticdrug resistance using SWCNT/methicillin.

Another aspect of the invention concerns the fact that it was alsopossible to visualize SWCNT fluorescent imaging inside S. epidermidisbacterial cells, thus demonstrating the ability to use SWCNTs as animaging tool to track drug delivery. The fluorescence imaging datasupport the potential use of this as a powerful research tool for drugdelivery and uptake, allowing further elucidation of antibioticresistance mechanisms.

As described herein, the systems and techniques of the invention presenta SWCNT antibiotic delivery system with demonstrated antibacterialactivity against Staphylococcus epidermidis using SWCNT/doxycycline andSWCNT/methicillin. As will be described in detail, the methods of theinvention have confirmed utility for two different classes ofantibiotics and quite notably, have shown efficacy in cells that areinitially resistant to methicillin, thereby suggesting an ability tobypass drug resistance via SWCNT-mediated delivery of methicillindirectly into the bacterial cells. Significantly, in the method of thepresent invention, nanotubes and antibiotics are complexednon-covalently, with no chemical bonding, but through adsorption ofantibiotics on the nanotube surface being governed by sufficientlystrong molecular attraction between hydrophobic systems of the two. Thistechnique allows antibiotics to get off the nanotubes easier as theyreach cell membrane. Otherwise water insoluble nanotubes are dispersedby antibiotics themselves.

Work was done with bacterial strains that, according to the experimentsdescribed herein, show resistance to antibiotics without nanotubes. Whenantibiotics are introduced with nanotubes, that resistance is mitigatedby nanotube transport potentially into the membrane of bacteria. Thestrains of S. epidermis used in the described experiments are the onlyones tested to date (due to safety issues) and are not the ones known tobe fully resistant (MRSE or MRSA). However, they are derivatives ofthose bacteria and show no response (resistance) to methicillinintroduced without nanotubes in 2 out of 3 tests conveyed. Thus,nanotubes help overcome that antibiotic resistance.

Additional objects, features and advantages will be apparent in thewritten description which follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(a) is a graph illustrating inhibition zones arising fromindividual components (SWCNT/DSPE-PEG 5000, Doxycycline, Methicillin)and SWCNT/antibiotic hybrids at two different concentrations;

FIG. 1(b) is an image of an inhibition zone of S. epidermidis treatedwith Doxycycline;

FIG. 1(c) is an image of an inhibition zone of S. epidermidis treatedwith SWCNT/Doxycycline;

FIG. 1(d) is an image of an inhibition zones of S. epidermidis treatedwith Methicillin;

FIG. 1(e) is an image of an inhibition zone of S. epidermidis treatedwith SWCNT/Methicillin.

FIG. 2(a) is a graph showing ANOVA and Dunnet's method statisticalanalysis of doxycycline disk diffusion data for 0.5 mg SWCNT, 2 mgantibiotic loaded;

FIG. 2(b) is a graph showing ANOVA and Dunnet's method statisticalanalysis of doxycycline disk diffusion data for 1 mg SWCNT, 4 mgantibiotic loaded;

FIG. 2 (c) is a graph showing ANOVA and Dunnet's method statisticalanalysis of methicillin disk diffusion data for 0.5 mg SWCNT, 2.5 mgantibiotic loaded;

FIG. 2(d) is a graph showing ANOVA and Dunnet's method statisticalanalysis of methicillin disk diffusion data for 1 mg SWCNT, 5 mgantibiotic loaded.

FIG. 3(a) is a graph of Colony Formation Unit assay of antibiotics andantibiotic-SWCNT dispersion. Colonies on each plate counted with OpenCFUsoftware.

FIG. 3 (b) is the S. epidermidis control;

FIG. 3(c) shows the S. epidermidis treated with Doxycycline;

FIG. 3(d) shows the S. epidermidis treated with Doxycycline-SWCNTdispersion;

FIG. 3(e) shows the S. epidermidis treated with Methicillin;

FIG. 3 (f) shows the S. epidermidis treated with Methicillin-SWCNTdispersion;

FIG. 4(a) is a graph of showing turbidity assay absorption data at 600 mfor Doxycycline and SWCNT/Doxycycline;

FIG. 4(b) is a graph showing turbidity assay absorption data at 600 mfor Methicillin and SWCNT/Methicillin;

FIG. 5(a) is a graph of Cytotoxicity assay in HeLa cells comparingresponse to Doxycycline vs SWCNT/Doxycycline;

FIG. 5(b) is a graph of Cytotoxicity assay in HeLa cells comparingresponse to Methicillin vs SWCNT/Methicillin;

FIG. 6 (a) shows the NIR Fluorescence imaging of SWCNT emission inbacterial cells subject to SWCNT/Methicillin treatment;

FIG. 6(b) shows the NIR Fluorescence imaging of SWCNT emission inbacterial cells subject to SWCNT/Doxycycline treatment;

FIG. 6(c) shows the NIR Fluorescence imaging of SWCNT emission inbacterial cells subject to Non-treatment control.

FIGS. 6(d) and 6(e) are SEM images of bacterial cells subject toSWCNT/methicillin hybrids.

DETAILED DESCRIPTION OF THE INVENTION

As discussed in the Background of the invention, due to the misuse andoveruse of conventional antibiotics, resistant infections are on therise. Those include well-known bacterial strains such as Staphylococcusaureus (MRSA) infection, Streptococcus pneumoniae, and Mycobacteriumtuberculosis. It is predicted that approximately 10 million people willdie from resistant infections by the year 2050 alone, not consideringthe emergence of new mutated strains. This mortality prediction exceedsthat of cancer and diabetes combined. In response to such a dauntingcrisis, the scientific community has been developing new antibiotics.However that approach is hardly sustainable and not long lived, rather,giving rise to multi-drug resistant infections such as MRSA and VRCE.With mutated bacterial infections being the foundation for manylarge-scale health issues including M. tuberculosis, MRSA and VRE, thecrisis of antibiotic resistance becomes a global threat.

To date very few routes were explored to address the antibioticresistance including the inhibition of mutation, change in dosingregimen of existing antibiotics and delivery-assisted combinationtreatments. However dosing strategies provide only temporary solutionthat delay the formation of resistance, and inhibitory approaches haveto be applied to bacteria prior to mutation that renders antibioticsineffective. With the few successful delivery-assisted attempts toaddress the existing antibiotic resistance, current studies still focusmore on the development of new antibacterial strategies. In the systemsand techniques of the present invention, an alternative multimodalapproach to the issue at hand is explored. Rather than developing anentirely new treatment, this work the first time proposes non-covalentlyformulating existing antibiotics with single-walled carbon nanotubes(SWCNTs) for delivery, imaging and enhanced antibiotic efficacy. SWCNTsprovide a new multifunctional route to antibiotic delivery withconcomitant capability of fluorescence-based tracking of transportedtherapeutics.

For the context of the studies which follow, the SWCNT-antibioticdispersions are tested against Staphylococcus epidermidis. Because ofthe increased use of biomaterials in the hospital and clinicalenvironment, S. epidermidis has become one of the five most commonbacteria to cause nosocomial infections on prosthetic parts, valves,surgical wounds, urinary tract or bone marrow transplants. Oncebacterial strains develop in these scenarios, it often difficult totreat them with antibiotics, forcing removal and replacement of theaforementioned tissues. While already causing nearly one millioninfections and many deaths per year, S. epidermidis has become resistantto a wide scope of antibiotics. Strains of S. epidermidis is resistantto methicillin, penicillin, penems, carbapanems, and cephalosporins.With these being the most commonly used antibiotics, an increase in S.epidermidis infections becomes a big threat.

SWCNTs offer great promise as antibiotic delivery vehicles due to theirunique physical and optical properties. Known for theirquasi-one-dimensional structure, SWCNTs have the dimensions suitable forcellular internalization show low cytotoxicity when formulated,accumulate partly in actin cytoskeleton but exhibit excretion over time.Additionally, a significant amount of SWCNTs can be loaded into a targetcell, making them suitable for the delivery of hydrophobic drugs andgene therapies sensitive to degradation in blood. SWCNTs also show apotential for antibiotic delivery as multiple antibiotics are known toadsorb well on SWCNT surfaces and with covalent attachment improve theefficacy of ciprofloxacin antibiotic. The mechanism of SWCNT interactionwith bacteria is so far unknown and can be further explored withmolecular imaging. SWCNTs can be used for that purpose as efficientbiomarkers since semiconducting species exhibit fluorescence in thenear-infrared where biological autofluorescence background is minimal.SWCNT emission does not experience photobleaching, avoiding one of thedrawbacks of conventional fluorophores, and can penetrate through thelayers of biological tissue due to low tissue absorption/scattering innear-IR. SWCNT-mediated fluorescence imaging can, therefore, be used totrack the location of therapeutics in the targeted cells and tissues.

Despite these advances in SWCNT molecular imaging and initial work onantibiotic transport, the issue of antibiotic resistance still remainsunsolved as there are no reports of SWCNTs recycling antibiotics towhich the bacteria were previously resistant and no knowledge of themechanism by which SWCNT delivery can address this resistance.Image-guided delivery of antibiotics allowing to track their transportand help elucidate SWCNT-guided mechanism of action has also not beenexplored to date as the covalent attachment of antibiotics is known toquench SWCNT emission.

The method of the present invention utilizes non-covalent SWCNTantibiotic delivery not only to enhance antibiotic efficacy and trackthe transport with intrinsic SWCNT fluorescence, but also to circumventthe antibiotic resistance of the bacteria previously showing resistantbehavior. The focus is on overcoming the lack of antibiotic sensitivityof S. epidermis to methicillin. Also, antibiotic resistance is basedpartly on enzymatic degradation of the existing antibiotics or adecreased membrane permeability to those. Using SWCNTs as deliveryvehicles, one aim of the present invention is to address both of theseissues as they are known to protect delivered gene therapeutics fromenzymatic degradation and enhance cellular internalization of other drugmoieties. Additionally other research has identified the antibacterialproperties of SWCNTs that may disrupt the membrane and/or metabolicprocesses and morphology of bacteria. This all suggests that SWCNTs maybe highly advantageous delivery vehicles for antibiotic treatment.

Due to their hydrophobic structure, in a number of biologicalapplications, SWCNTs are dispersed via biocompatible surfactantsincluding PEG or PEI derivatives. Although those mask SWCNTs in blood,yielding longer circulation times, additional dispersing agents increasethe complexity of the formulation potentially hampering the release ofthe therapeutic. This work uses a unique antibiotic delivery methodfunctionalizing SWCNTs non-covalently with antibiotics themselvesallowing for SWCNT dispersion in aqueous media and antibiotic deliverydirectly on the SWCNT surface. Such non-covalent delivery also improvesthe possibility of antibiotic release within bacterial cells.

Using SWCNTs in combination with existing antibiotics provides asynergetic approach which, using the principles of the invention, yieldsnot only successful delivery and imaging, but up to 70% improvement ofthe antibiotic efficacy for antibiotics to which bacteria showedresistant behavior. This may open a novel route for recycling existingantibiotics and effectively combating antibiotic resistance, the majorissue in antibacterial treatment.

Methods 1.1 Dispersion of SWCNT in Antibiotic Solutions

Aqueous Doxycycline (20 mg/ml) and Methicillin (25 mg/ml) antibioticsolutions were used in this study to complex with SWCNT. Various SWCNTand antibiotic concentrations were tested to obtain an optimalconcentration of SWCNTs of 500 μg dispersed in 1 mL of the correspondingantibiotic solution This yields the highest fluorescence efficiencies of−1 representing the ratios of integral fluorescence to integralabsorption assessed using Applied Nanofluorescence NS-2Nanospectralyzer. Each antibiotic in aqueous solution was complexed with500 μg of raw HiPco (Nanointegris batch #HR27-075A) non-covalently via30 min of ultrasonic bath treatment followed by 20 min ultrasonic tiptreatment at 3 W of power. SWCNT/antibiotic dispersions were centrifugedfor 30 min at 16000×G to remove SWCNT aggregates. Resulting suspensionscontaining individual antibiotic-suspended SWCNTs were characterized viaabsorption spectroscopy and stored at 4° C. with further exposure to 2min ultrasonic treatment prior to use.

For control experiments solution of SWCNTs/DSPE-PEG 5000 was prepared:0.5 mg of SWCNT was added to a 1600 μM solution of DSPE-PEG 5000(NanoCS) and subjected to the aforementioned ultrasonic dispersion andfiltration procedures to yield final SWCNTs/DSPE-PEG 5000 suspensions.

1.2 Characterization of SWCNT-Antibiotic Dispersions

Concentration of all SWCNT suspensions were characterized via absorptionspectroscopy. Using standard calibration curve constructed fromabsorptions of unfiltered SWCNT/antibiotic fractions with known SWCNTamounts we have experimentally derived extinction coefficients at 632 nmfor SWCNT dispersed with both drugs (0.015 (μg/mL)⁻¹ forSWCNTs/doxycycline, and 0.0134 (μg/mL)⁻¹ for SWCNTs/methicillin). Wefurther used those to assess the concentration of SWCNTs in centrifugedsuspensions.

Concentration of antibiotic in the suspensions of antibiotic/SWCNThybrids was assessed via deconvoluting absorption spectra of those intocomponents for SWCNTs and antibiotic. SWCNTs/DSPE-PEG 5000 spectra wereused as an assessment for SWCNT component and antibiotic standards atknown concentrations were used as reference component for antibiotics.This calculation showed w/w ratios of 1:4 for SWCNT/methicillin and 1:5and SWCNT/doxycycline in stock SWCNT suspensions that were further usedthroughout this work.

1.3 Disk Diffusion Assay

S. epidermidis broth of McFarland 0.5 standard (absorption of 0.08 to0.1) was created using Mueller Hilton Broth. This standard stabilizedthe cell count at an approximate 1×10{circumflex over ( )} 8 CFU/mL.Dilution was plated within 15 minutes of standardization. Following theproper aseptic techniques, 0.2 mL of bacterial broth was placed in thecenter of prepared agar dish. A sterile bacteria spreader was used toevenly spread the bacteria throughout the plate to create a lawn.

Two different dosages of the antibacterial solutions were tested toincrease the breadth and reliability of data. Blank sensitivity discswere loaded with 10 μL and 20 μL (based on respective dosage) of stocksuspensions and placed onto the surface of the agar using sterileforceps. Discs were impregnated with the test solution dropwise. Fivediscs were evenly placed equidistant from one another. Before tiltingover the Petri dishes, discs were left to dry and gently pressed down toensure attachment to agar. Once all Petri dishes are prepared, they wereturned upside down to prevent surface condensation. Petri dishes areincubated for 24 h at 37° C., then the zones of inhibition were measuredwith the inclusion of disk diameter in the measurements.

1.4 Colony Count Assay

Using S. epidermidis broth (McFarland 0.5 standard), 100 μl was placedin the center of the agar plate. 100 μl of the respective antibacterialstock solution is added to the center. Bacteria was spread through thePetri dish and the plates were further incubated for 24 h at 37° C.Pictures of the plates were uploaded onto the OpenCFU software to countthe number of colonies grown on the plate. Two plates were prepared foreach antibacterial treatment with corresponding controls.

1.5 Turbidity Assay

A serial dilution (using the factor of 2) of antibacterial solutions wasconducted in 12-well plates starting with 200 μL of antibacterialsolution was placed in first well. 850 μl of broth and 50 μl ofbacterial broth were added to each well plate. The concentrations testedfor doxycycline were 1, 0.5, 0.25, 0.125 and 0 mg/mL. The concentrationstested for methicillin are 1.25, 0.625, 0.313, 0.106 and 0 mg/mL. Plateswere then incubated at 37° C. for 24 h. The solutions were transferredto cuvettes, and their turbidity is measured using a Cary 500spectrophotometer with the broth used as a baseline. Two wells wereprepared for each concentration.

1.6 Cytotoxicity Assay

An MTT cytotoxicity assay was conducted for 4 samples: doxycycline,SWCNTs/doxycycline, methicillin, and SWCNTs/methicillin), Thiazolyl BlueTetrazolium Bromide, and DMSO. Each sample was prepared via serialdilutions at the testing concentrations ranging from 0 to 3.5 μg/mL fordoxycycline and SWCNTs dispersed with doxycycline and 0 to 0.25 μg/mLfor methicillin and SWCNTs dispersed with methicillin. The absorbance ismeasured using the FLUOstar Omega microplate reader, and analyzed usingOmega software.

1.7 Microscopy

We utilize InGaAs near-IR (NIR) camera coupled to hyperspectralfluorescence filter (Photon etc.) to perform fluorescence microscopy ofSWCNTs imaged in bacterial cells 24 h after introducing to bacterialculture. The sample was excited with 637 nm diode laser excitation at700 mW output power. SWCNTS showed up in the nIR broadband (900-1450 nm)images (FIG. 6) as bright fluorescent objects. Non-treatment controlimages were taken for each antibiotic target ensuring no emission in thenear-IR. Scanning Electron Microscope (JEOL-JSM-7100F) was used at 5 kVto image bacterial cells deposited from the culture onto conductivecarbon tape. SEM allowed imaging the outer surface of bacterial cellsand extracellular SWCNTs.

Results & Discussion 2.1 Characterization

Noncovalently complexed SWCNT/antibiotic hybrids were prepared in thiswork for the first time. Antibiotics were utilized both as a payload andas surfactant for SWCNTs providing stable aqueous dispersions withfluorescence efficiencies remaining high (close to 1) for over a week.Doxycicline and methicillin were specifically chosen for this role dueto characteristic hydrophobic regions in their structure some of whichare expected to non-covalently bind to SWCNTs via π-stacking. Whiledoxycycline is a tetracycline antibiotic and inhibits reproduction bydisrupting protein synthesis, methicillin is a beta-lactam antibioticthat affects the bacteria by interfering with membrane structure. Usingtwo antibiotics from different classifications allows for anunderstanding of how SWCNTs perform with different modes of action.Additionally, S. epidermis strain used in this work shows no/lowresponse to methicillin suggesting some resistance and thus is intendedas a control for the studies.

As SWCNTs alone are insoluble in water, their successful dispersionhelps verify their complexation with antibiotics. Characteristicabsorption spectra of SWCNTs and antibiotics were used to assessconcentrations of those upon complexation. As absorption of bothantibiotics is negligible in the visible, the value of absorption at 632nm is used to determine the amount of SWCNTs (FIGS. 4(a) and (b)). Acalibration curve constructed with known concentration of SWCNTs inunfiltered suspensions of SWCNT/antibiotic hybrids allows to determineextinction coefficients for SWCNT/doxycycline and SWCNT/methicillinabsorption in the visible to be 0.015 (μg/mL)⁻¹ and 0.0134 (μg/mL)⁻¹respectively. Antibiotic concentration in the final dispersion isassessed by deconvoluting spectra of antibiotic/SWCNT hybrids viapresenting those as a superposition of those of SWCNT and antibioticalone at known concentrations, which resulted in the w/w ratios of −1:4for SWCNT/methicillin and −1:5 and SWCNT/doxycycline aqueoussuspensions.

2.2 Antibacterial Performance of SWCNT-Antibiotic Dispersions

Three different antibacterial sensitivity assays were used to verify theefficacy of SWCNT/antibiotic dispersions. The disk diffusion assay,colony formation assay and turbidity assay confirm the findings of theinvention through three different procedures.

2.2.1 Zone Inhibition Assay

The antibacterial effects of pure antibiotic solutions are firstcompared to non-treatment control and vehicle control provided bySWCNT/PEG formulations. Here DSPE-PEG-5000 is used as a biocompatibledispersing agent for SWCNTs to provide stable aqueous dispersionswithout the use of antibiotics.

In this formulation SWCNTs show little to no antibacterial effect, whichis supportive of their sole role as drug delivery/imaging agentsproviding no interference with antibacterial efficacy of the payload.Among the antibiotics, doxycycline is significantly more effective atboth 2 and 4 mg doses, whereas methicillin shows no antibiotic activitywith inhibition levels same to non-treatment control. Since strains ofS. epidermis are known to show antibiotic resistance and several arespecifically resistant to methicillin. The complete lack of inhibitionresponse with this antibiotic for colony formation and disc diffusionassays likely indicates a resistant behavior of the current strain tomethicillin. At the same time no resistance to doxycycline has beenobserved.

Concentrations of antibiotics in complexes with SWCNTs were furtherchosen to mimic those of unformulated antibiotics. SWCNT/antibioticdispersions show substantial improvement in the inhibition response forboth antibiotics. SWCNT/Doxycycline dispersion becomes slightly moreeffective than Doxycycline alone (8% improvement), whereasSWCNT/Methicillin hybrids exhibits a drastic improvement: an increase inefficacy from no observable bacterial inhibition to the 50% of theinhibition response of doxycycline. Because S. epidermidis in this testis initially resistant to methicillin, this increase in antibacterialefficacy signifies that SWCNT delivery is likely to bypass antibioticresistance. While there are a number of theories regarding themechanisms of cellular internalization of carbon nanotubes, many areproposing a more efficient entry due to interaction of hydrophobic SWCNTplatform with membrane lipids. Depending on their surface properties,SWCNT may partially disrupt the membrane leading to its portion orpotentially cause oxidative stress-mediated damage to bacterialmembranes. These mechanisms of nanotube-membrane interaction at higherconcentrations may lead to antibacterial effects. Thus, considering thatmethicillin's mechanism of action is based on the disruption ofbacterial cell membranes it is plausible that SWCNT/membrane interactionmay prevent the recognition of antibiotic by the resistant bacteria, andfacilitate its delivery inside the membrane where methicillin maysuccessfully perform its primary function.

2.2.2 Statistical Analysis

The statistical analysis performed in this work confirm the significanceof the observed results for diffusion discs performed at differentamounts of SWCNT/antibiotic complex loaded onto discs. Data analysis wasperformed using IMP software utilizing ANOVA (Analysis of Variance) toprovide the statistical information to understand the predominanteffects and significance of differences in data. In performing an ANOVAon a dataset, a null hypothesis was made, stating that the average meansof the various treatments are the same. When the p-value of the analysisis lower than the confidence level chosen, the hypothesis is provenfalse and a significant difference and variation is detected.

The dataset is analyzed using a comparison of means function byassigning a mean of the control group and comparing it to the means ofeach of the treatment groups. The degree of overlap of the circles inthe graphic represents their significant similarity or difference,whereas the size is proportional to the corresponding variance.

For comparison of antibiotic effects of SWCNT/Doxycicline to the freeantibiotic (FIGS. 2(a), (b)), the differences in data are significant.For both dosages of doxycycline the R-square is fairly high (˜0.97), andthe Prob>F is low (<0.0001), indicating a considerably good fit of thedata. Based on the Control Dunnett's method, the difference between thecontrol (antibiotic alone) and SWCNT/antibiotic hybrids is insignificantfor 2 mg dosage, but is considered significant for 4 mg dosage. Overall,it can be inferred that the complexes of SWCNT/doxycycline exhibitmarginally better activity than antibiotic alone. Unlike in the case ofdoxycycline, statistical analysis shows a significant improvement inantibacterial efficacy for SWCNT/methicillin hybrids. R-squares arerelatively high (˜0.9 and 0.8) and prob>F are low (<0.0001), indicatinga good fit of the data. The Control Dunnet's circles for both doses(FIGS. 2 (b),(c)) show that the difference between control and treatmentis significant and SWCNT/methicillin complex is statistically much moreefficacious than methicillin alone.

The results from the colony formation assay support that the efficacy ofSWCNT/antibiotic hybrids is greater than that of the antibiotics alone.SWCNT/Doxycycline formulation provides slightly lower colony counts thanthe antibiotic alone with 68% improvement both being highly effectiveagainst S. epidermis. The colony count for methicillin alone is similarto the control, as expected, whereas that for SWCNTs/methicillin issuppressed 40-fold (4000% improvement) showing significant observable(FIG. 3f ) decrease in the number of colonies. Such drastic increase inefficacy for SWCNT-delivered methicillin with corresponding only minorimprovement for already effective doxycycline can be likely attributedto bypassing of antibiotic resistance via SWCNT delivery.

The turbidity assay performed with SWCNT/antibiotic formulations inbacterial media assessed scattering from turbid samples proportional tobacterial concentration in suspension. Relative scattering is assessedby the magnitude of scattering background in absorption spectra sampledat 600 nm. Due to the low concentrations of antibiotics and SWCNT usedin this study, SWCNT absorption does not interfere with turbiditymeasurements. The absorption data in FIGS. 4a and 4b is thereforepresented as a Minimum Inhibitory Concentration Test. Rather than usinga MIC value however, the overall turbidity curve is utilized to assessthe amount of bacteria remaining in the respective suspensions. Asconsistent with findings of two previous methods, bacteria treated withSWCNT/methicillin complexes exhibit a lower turbidity than the samedoses of methicillin alone. Although antibiotic control for this testshows some bacterial inhibition, a significant improvement upon SWCNTcomplexation is noted. Doxycycline/SWCNTs hybrids yield slightly lowerturbidity in bacterial media as opposed to antibiotic alone as opposedto significant improvement for Methicillin.

As seen in all of the bacterial sensitivity assays, the complexation ofantibiotics with SWCNTs increases the efficacy of the treatment whencompared to antibiotic alone. SWCNT in this role may act as efficientdrug carriers or, potentially, enhance effect of antibiotics in acombination treatment. While the improvement of doxycycline efficacy viathe dispersion with SWCNTs is minimal, SWCNT/Methicillin complexesbecome far more efficacious bypassing the antibiotic resistance tomethicillin.

2.4 Cytotoxicity

The observed effect was verified as not being due to inherent SWCNTtoxicity in complexation with methicillin by a separate MTT cytotoxicityassay in HeLa cells (FIG. 5). For both Doxycycline and MethicillinSWCNT/antibiotic complexes at the same antibiotic dose show lessinherent toxicity to HeLa cells than antibiotics alone indicating notoxic effect from SWCNTs.

2.5 Imaging

The inherent emission of SWCNTs in the near-IR was utilized to track andimage those in bacterial cells collected from bacterial culture aroundthe discs in the disc diffusion assay to verify cell internalization.Control bacteria were imaged together with the ones subject toSWCNT/methicillin and SWCNT/doxycycline treatments loaded on the discs.As biological autofluorescence background is minimal in the near-IR, weexpect SWCNTs to be the major emissive species. In accordance with this,control cells show no observable emission whereas microscopy images ofbacterial cells recovered in the vicinity of the discs withSWCNTs/methicillin and SWCNTs/doxycycline show bright near-IR SWCNTemission in the clusters bacterial cells. The highest signal intensitieswere found surrounding cells or cell clusters suggesting possibleincorporation into the membrane. Extracellular SWCNT emission appears tobe rare indicating preferential interaction of SWCNT/antibiotic hybridswith bacteria.

Considering resolution limitations of fluorescence imaging of smallmicrometer-sized bacterial cells, these findings were confirmed by thehigher resolution SEM imaging of bacteria subject to SWCNT/methicillinhybrids. In SEM images showing the outer surface of the bacteria SWCNTare clearly observed associating with the membrane of bacterial cells inlarge quantities with some incidences of membrane penetration caught inthe image. This preferential accumulation is likely to result in theenhanced antibiotic effect observed with SWCNT/doxycycline hybrids asits mechanism of action through inhibiting bacterial protein synthesismay be positively affected by the amount of the drug. As formethicillin, SWCNT-assisted penetration into cell membrane may aid theantibiotic to bypass the need to bind to PBP membrane receptors. S.Epidermis as well other strains expressing mutated form of PBP2 withlower binding affinity and increased release rates which suppressesmethicillin membrane attachment and thus inhibits its efficacy.

Experimental results observed here suggest that SWCNTs acting asdelivery vehicles with no inherent antibacterial activity provide analternative route to membrane localization.

3. Conclusion

This work for the first time explores the joint delivery and imaging ofantibiotics by single-walled carbon nanotubes. SWCNTs dispersed in waterwith doxycycline and methicillin non-covalently attached to theirsurface act as drug delivery vehicles facilitating the improvedantibacterial effect in Staphylococcus epidermidis. In three differentsensitivity assays performed in this work, the advantages of aSWCNT/antibiotic therapy are apparent. SWCNTs facilitate preferentialbacterial accumulation of the payload and improved antibacterial effectfor antibiotics of two different classes. A relative improvement withrespect to antibiotic treatment alone varied from 68% increase inbacterial colony inhibition observed for SWCNT/doxycycline hybrids to40-fold (4000%) improvement in bacterial colony inhibition for SWCNTcomplex with methicillin, to which S. epidermidis initially showsresistant behavior in our assays. These results confirmed bystatistically significant findings from disc diffusion and a generaltrend provided by the turbidity assays suggest that whereas fordoxycycline enhanced efficacy can be explained by increased uptakefacilitated by SWCNT delivery; SWCN/methicillin complexes are likelybypass the antibiotic resistance of S. epidermidis. Based on thereported interaction of SWCNTs with cellular membrane, direct transportof the antibiotic into the membrane affected by SWCNTs appears to occurwhere methicillin's mechanism of action becomes effective or masking itsrecognition via SWCNT delivery. These hypotheses are supported by SWCNTfluorescence imaging within bacterial cell culture subject toSWCNT/antibiotic treatment indicating substantial SWCNT fluorescencesignal originating from bacteria rather than extracellular environment.SEM images confirm the association of SWCNTs with the membrane ofbacteria.

In this work SWCNT act as effective multifunctional antibioticdelivery/imaging agents with the potential to bypass antibioticresistance. As methicillin is one of the more widely known antibioticsfor developing resistance, its activation through noncovalenthybridization with SWCNTs offers an alternative potential approach tothe antibiotic resistance crisis. It may further provide a chance toreduce the dose, reuse and recycle the existing antibiotics for thetreatment of the new resistant bacterial epidemics.

While the invention has been shown in several of its forms, it is notthus limited but is susceptible to various changes and modificationswithout departing from the spirit thereof.

What is claimed is:
 1. A method for treating a bacterial infection, themethod comprising the steps of: providing a single-walled carbonnanotube comprising a nanotube surface; complexing an antibiotic withthe single-walled carbon nanotube to form a nanotube/antibiotic hybrid;and administering the nanotube/antibiotic hybrid; wherein the antibioticis delivered to a bacterial cell.
 2. The method of claim 1, wherein theantibiotic is complexed to the single-walled carbon nanotube viaadsorption of the antibiotic onto the nanotube surface.
 3. The method ofclaim 2, wherein adsorption of the antibiotic onto the nanotube surfaceis facilitated by interaction between a hydrophobic region of theantibiotic and a hydrophobic region of the single-walled carbonnanotube.
 4. The method of claim 1, wherein the antibiotic is selectedfrom the group consisting of doxycycline and methicillin.
 5. The methodof claim 1, further comprising the step of dispersing the single-walledcarbon nanotube and antibiotic in water such that the antibiotic isadsorbed onto the nanotube surface.
 6. The method of claim 1, whereinthe bacterial cell is resistant to the antibiotic.
 7. The method ofclaim 1, wherein the bacterial cell is Staphylococcus epidermidis. 8.The method of claim 1, wherein delivery of the antibiotic to thebacterial cell comprises cellular internalization of the antibiotic bythe bacterial cell such that an antibiotic resistance mechanism of thebacterial cell is circumvented.
 9. The method of claim 1, furthercomprising the step of causing the single-walled carbon nanotube ornanotube/antibiotic hybrid to exhibit fluorescence.
 10. The method ofclaim 1, further comprising the step of visualizing the single-walledcarbon nanotube or nanotube/antibiotic hybrid after administration viafluorescence imaging.
 11. The method of claim 9, further comprising thestep of using the fluorescence to track the single-walled carbonnanotube or nanotube/antibiotic hybrid.
 12. A method for introducing aselected antibiotic drug to an organism using a nanotube for thetherapeutic purpose of treating a bacterial infection, the methodcomprising the steps of: providing a single-walled carbon nanotubehaving a nanotube surface as a substrate for the selected antibioticdrug; wherein the selected antibiotic drug is complexed to the substrateto form a nanotube/drug hybrid by non-covalently bonding the antibioticdrug to the nanotube surface through adsorption of the antibiotic ontothe nanotube surface, the bonding being governed by sufficiently strongmolecular attraction between hydrophobic systems of antibiotic drug andthe carbon nanotube; and administering the nanotube/drug hybrid; whereinthe antibiotic is delivered to a bacterial cell.
 13. The method of claim12, wherein the antibiotic drug is selected from the group consisting ofdoxycycline and methicillin.
 14. The method of claim 12, wherein thesingle-walled carbon nanotubes are dispersed in water with a selectedone of the doxycycline and methicillin so that the selected antibioticdrug is non-covalently attached to the nanotube surface.
 15. The methodof claim 12, wherein the delivery device is effective in delivering anantibiotic drug which achieves an improved antibacterial effect againstStaphylococcus epidermidis.
 16. The method of claim 12, wherein themethod is used for cellular internalization of the selected antibioticand acts as a method for circumventing bacterial resistance to theselected drug.
 17. The method of claim 12, further comprising the stepsof: visualizing the single-walled carbon nanotube fluorescence imaginginside the bacterial cells; using the fluorescence imaging data as animaging tool to track drug delivery.